Project Summary Diabetes mellitus affects over 400 million people worldwide. The majority of this affected population is diagnosed with type 2 diabetes (T2DM). Recent clinical studies have demonstrated that patients with T2DM are at higher risk than non-diabetic patients for Parkinson?s disease (PD) and shared subcellular pathologic features indicate that these disorders have common mechanistic underpinnings. Clinical trials have begun to investigate the therapeutic benefit of various T2DM treatments in the context of PD. A new generation of insulin sensitizers engineered to inhibit mitochondrial pyruvate carriers (MPCs) has shown therapeutic promise in experimental models of T2DM and PD. As MPCs are a drug target in the treatment of both disorders, further study of these transmembrane proteins could uncover a mechanistic link between T2DM and PD. MPCs are highly conserved between yeast and humans and therefore this study proposes to take advantage of the simplicity and genetic malleability of the model organism Saccharomyces cerevisiae. This project will provide a deeper understanding of the role of MPCs in regulating cellular metabolism, organelle dynamics, and mitophagy. My first aim will investigate the hypothesis that MPCs are responsible for the transport of branched- chain amino acid (BCAA) metabolites, specifically ?-ketoisovalerate (KIV). To achieve this goal I will first engineer yeast strains with altered MPC monomer expression. Isolated mitochondria from these strains will be subjected to biochemical assays and gas chromatography instrumentation will be used to determine the resulting substrate and product concentrations. The second hypothesis investigated by this study is that the lack of functional MPCs will increase mitochondrial tethering to the endoplasmic reticulum and vacuole within yeast. To evaluate intracellular organelle dynamics and morphology I will employ fluorescent reporters and microscopy techniques. The third aim will explore the hypothesis that MPC inhibition decreases mitochondrial recycling and ATP production. Mitochondrial degradation and ATP production will be investigated by employing imaging, immunoblotting, and respiration assays. This project will clarify the downstream effects of MPC inhibition, thereby helping to uncover the molecular basis for the link between T2DM and PD. By providing a better understanding of the impact of MPC inhibition on cellular metabolism, organelle dynamics, and mitochondrial function, this study will inform the development of novel therapeutics for both disorders. The proposed research project will be conducted at Princeton University under the guidance of a superbly suited team of mentors (Sponsor: Dr. Jos Avalos, Co-sponsor: Dr. Coleen Murphy, Collaborators: Dr. Clifford Brangwynne and Dr. Daniel Cohen). The enclosed proposal contains a training plan to improve knowledge of scientific techniques, enhance critical thinking, and refine communication of scientific material. Additionally, this plan provides opportunities for clinical continuity. Each component of this study was crafted to provide a solid foundation for an independent research career as a physician-scientist.